WO2012087153A1 - Enrichissement d'huiles d'animaux marins en acides gras oméga 3 polyinsaturés par une hydrolyse catalysée par une lipase - Google Patents

Enrichissement d'huiles d'animaux marins en acides gras oméga 3 polyinsaturés par une hydrolyse catalysée par une lipase Download PDF

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WO2012087153A1
WO2012087153A1 PCT/NO2011/000354 NO2011000354W WO2012087153A1 WO 2012087153 A1 WO2012087153 A1 WO 2012087153A1 NO 2011000354 W NO2011000354 W NO 2011000354W WO 2012087153 A1 WO2012087153 A1 WO 2012087153A1
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oil
omega
fatty acids
hydrolysis
lipase
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PCT/NO2011/000354
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Kahveci DERYA
Xu XUEBING
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Marine Bioproducts As
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Publication of WO2012087153A1 publication Critical patent/WO2012087153A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/158—Fatty acids; Fats; Products containing oils or fats
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/12—Refining fats or fatty oils by distillation
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/16—Refining fats or fatty oils by mechanical means
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C1/00—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids
    • C11C1/02—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils
    • C11C1/04—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils by hydrolysis
    • C11C1/045—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils by hydrolysis using enzymes or microorganisms, living or dead
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6418—Fatty acids by hydrolysis of fatty acid esters

Definitions

  • the present invention relates to a method for enrichment of omega-3 polyunsaturated fatty acids comprised in acylglycerols of an oil by lipase-catalysed hydrolysis as well as to a processed marine oil and the use of the processed marine oil.
  • Marine oils are generally recognized as being beneficial and/or preventive to human and animal health due their high content of polyunsaturated fatty acids (hereinafter "PUFA"), especially the omega-3 fatty acids eicosapentaenoic acid (20:5 n-3; EPA) and docosahexaenoic acid (22:6 n-3; DHA).
  • PUFA polyunsaturated fatty acids
  • Omega-3 fatty acids eicosapentaenoic acid (20:5 n-3; EPA) and docosahexaenoic acid (22:6 n-3; DHA).
  • Positive effects of omega-3 fatty acids have been reported for numerous conditions such as cardiovascular diseases, atherosclerosis, several types of cancer, dyslipidemia, hypertension, diabetes, obesity, inflammatory diseases, neurological/neuropsychiatric disorders, asthma and rheumatoid arthritis.
  • DHA being a main component of nervous tissue
  • EPA being a main component of nervous tissue
  • Beside other effects feeding of DHA has been shown to result in a significant reduction in blood pressure and heart rate.
  • EPA on the other hand, being quantitatively the main omega-3 PUFA compound, has anti-inflammatory effects due to its regulatory role in gene expression.
  • a recent meta-analysis revealed that significant improvement of mood in patients with depression was obtained
  • omega-3 fatty acids in oils are of importance for its later value and application but also the quantitative composition of the comprised omega- 3 fatty acids as well as their ratio i.e. EPA/DHA.
  • Oils comprising omega-3 PUFA are frequently extracted from wild caught marine resources such as from fish and krill.
  • marine oils can also be obtained from farmed marine organisms such as farmed fish.
  • the use of plant oils of terrestrial origin substantially not comprising any omega-3 fatty acids has increased in feed for aquaculture production, especially for Salmon ids such as the Atlantic salmon. Since salmon is dependent on receiving these fatty acids with their diet, fish fed a low amount of omega-3 fatty acids will also store a reduced content of these fatty acids in their lipids.
  • Oils obtained from these fish will consequently have a lower omega-3 fatty acid content, which reduces the value of these oils for applications such as for human and animal consumption, pharmaceutical compositions, functional feed and health products. This is one major concern of the fish farming industry when making efficiently use of those oils from farmed fish. Application of oils obtained from farmed animals are preferred on the other hand due to their in general low degree of contamination with environmental pollutants and their stable quality compared to oils obtained from wild catches.
  • PUFA omega-3 polyunsaturated fatty acids
  • the available methods for concentrating/enrichment of PUFA include adsorption chromatography, fractional or molecular distillation, enzymatic splitting, low-temperature crystallization, supercritical fluid extraction and urea complexation. Only few of these methods are suitable for large-scale processes and each of these techniques is recognised for its own advantages and draw backs.
  • Enzymatic processes for concentrating omega-3 fatty acids were previously recognised as particularly advantageous when handling PUFA, since these fatty acids are highly sensitive to oxidation. Enzymatic methods allow the application of mild reaction conditions, meaning lower temperature and pressure, which is important when dealing with omega-3 fatty acids. Low temperature also improves the feasibility of the process. Furthermore, enzymatic processes are considered as more environmentally friendly compared to chemical ones.
  • lipase-catalysed hydrolysis is one of the most widely used enzymatic reaction, for the purpose of improvement of the omega-3 concentration of fish oil.
  • the lipase catalysed hydrolysis the amount of omega-3 fatty acids in the acylglycerols is increased (enriched) in relation to the total amount of fatty acids comprised in the acylglycerols.
  • the key property of the lipase-catalysed process lies in the fatty acid (FA) selectivity of lipases, since most of them discriminate against PUFA and preferably hydrolyse saturated and monounsaturated fatty acids (SFA and MUFA) bound in the
  • acylglycerols Saturated and monounsaturated fatty acids are thus released as free fatty acids during the lipase-catalysed hydrolysis. Thereby the amount of PUFA fatty acids increases in the acyl glycerol fraction in respect to the total amount of fatty acids present in the acyl glycerol fraction.
  • the enzymatic process is commonly followed by a separation process such as membrane filtration or molecular complexation whereby the hydrolysed free fatty acids are removed and an oil is obtained which has an acylglycerols fraction with an increased/enriched content of omega-3 fatty acids.
  • Lipase-hydrolysed concentrating of oils are described in the prior art. However, the efficiency of the disclosed processes and thus their applicability in industrial scale is often not satisfactory.
  • the selectivity of the lipases decreases typically during the process since the availability of the SFA and MUFA eventually decreases, leading to a loss of PUFA e.g. of EPA.
  • Carvalho et al. Enzymatic hydrolysis of Salmon Oil by native lipases: Optimization of process parameters, J. Braz. Chem. Soc, 2009, Vol 20, No.
  • Candida rugosa also referred to as Candida cylindracea
  • Candida rugosa is one of the enzymes which has been used in concentrating of
  • the obtained glyceride mixture is hydrolysed by a lipase from a Penicillium- derived lipase, that does not hydrolyse triglycerides to obtain a glycerides fraction with high DHA amounts.
  • the aim of the present invention is to provide a simple, mild, efficient and at the same time highly selective method for the improvement of fish oils by lipase- catalysed hydrolysis, which can be used in an industrial large scale in order to obtain omega-3 PUFA at higher yield and purity at lower cost than the existing methods. Furthermore, the invention also aims at providing a method for the concentration of omega-3 PUFA in oils wherein the content in EPA and DHA is balanced in the final product.
  • the present invention concerns a method for enrichment of the amount of omega-3 polyunsaturated fatty acids comprised in acylglycerols of an oil by lipase-catalysed hydrolysis, wherein the oil is mixed with an aqueous solution in a water to oil ratio of 2:1 to 5:1
  • a lipase which selectively hydrolyses saturated and monounsaturated fatty acids bound to said acylglycerols and discriminates in its hydrolytic activity against omega-3 polyunsaturated fatty acids bound to said acylglycerols, is added to the oil- water mixture in a concentration of 3-5.5 % by weight based on the amount of oil.
  • the free fatty acid are separated from the acylglycerol fraction after the hydrolysis, and the lipase-catalysed hydrolysis of the separated acylglycerol fraction is repeated. More preferably, the same enzyme is used in the first and second hydrolysis step of the repeated hydrolysis.
  • the lipase is a microbial lipase, preferably from a species selected from the group consisting of Pseudomonas, Candida, Rhizopus, and Rhizomucor.
  • the lipase is selected from the group consisting of Candida rugosa, Burkholderia cepacia, and Rhizopus oryzae.
  • the incubation temperature in the lipase-catalysed hydrolysis is in the range of about 30-50°C, more preferably of about 40 to 50 °C, and most preferably of about 45 °C.
  • the incubation time in the lipase-catalysed hydrolysis is between 2 hours and 8 hours, more preferably between 2 and 4 hours.
  • omega-3 polyunsaturated fatty acids are chosen from DHA, EPA and/or DPA.
  • said lipase discriminates in its activity between EPA and DHA.
  • the hydrolysis conditions are as follows
  • the water-to-oil ratio is in the range of 2:1 to 5:1 (v/w),
  • the incubation temperature is in the range of about 30-50°C
  • the incubation time is between 2 hours and 4 hours. More preferred the lipase is added in a concentration of about 3 % by weight based on the amount of oil, and the hydrolysis conditions are as follows
  • the incubation temperature is about 45°C
  • the incubation time is between 2 and 4 hours.
  • the lipase is added in a concentration of about 3-4 % by weight based on the amount of oil, and the hydrolysis conditions are as follows
  • the incubation temperature is about 30°C
  • the water-to-oil ratio is in the range of about 2.5-5:1 (v/w),
  • said oil to be enriched with omega-3 polyunsaturated fatty acids which are comprised in the acylglycerols is a marine oil obtained from a fish, a crustacean, a bacterium, a macroalgae and/or a microalgae.
  • the oil is obtained from one or several fish species selected from the group consisting of Salmonids, Gadoids, Clupeids, Engraulidae, Scromboids, and Elasmobranchs, which can be wild caught or farmed fish.
  • Salmonids Gadoids, Clupeids, Engraulidae, Scromboids, and Elasmobranchs, which can be wild caught or farmed fish.
  • said oil is from a farmed marine animal comprising a reduced amount of omega-3 polyunsaturated fatty acids compared to an oil obtained from the same species in the wild.
  • the oil has a polyunsaturated fatty acid content of no more than 16 % mol, preferably of no more than 14 % mol, at the start of the enrichment process with polyunsaturated omega-3 fatty acids.
  • the acylglycerol fraction comprising the enriched omega-3 polyunsaturated fatty acids is separated from the free fatty acids after the enzymatic hydrolysis, preferably by use of short path distillation.
  • the oil phase is fed to a short path distillation unit at a rate of 2 mLJmin under 10 3 mbar of vacuum, wherein the feeding tank is set at 35°C, the condenser at 45°C, and the evaporator at 145°C.
  • the present invention relates to processed marine oil wherein the amount of polyunsaturated fatty acids in the acylglycerols of said oil has been enriched by a method according to any of the preceding paragraphs.
  • the present invention relates to a processed marine oil wherein the content of omega-3 polyunsaturated fatty acids in the acylglycerols has been enriched characterised in that at least 33 mol % of the fatty acids comprised in the acylglycerols are omega-3 polyunsaturated fatty acids and the EPA/DHA ratio is 0.4 or more.
  • At least 55 mol % of the fatty acids comprised in the acylglycerols are omega-3 polyunsaturated fatty acids and the EPA/DHA ratio is 0.3 or more.
  • the oil is a fish oil, preferably from a farmed salmonid, more preferred from a farmed salmon.
  • the oil has an omega-3-PUFA content of less than 18 % before enrichment of the acylglycerols with omega-3 PUFA and has an omega-3- PUFA content of more than 36 % after the enrichment, preferably of more than 48 %. It is also preferred that the oil has a DHA content of less than 10 % before enrichment of the acylglycerols with omega-3 PUFA and has a DHA content of more than 20 % after the enrichment, preferably of more than 30 %.
  • the oil has an EPA content of less than 7 % before enrichment of the acylglycerols with omega-3 PUFA and has an EPA content of more than 8 %, preferably of more than 10 % after the enrichment.
  • the oil is from a pelagic fish, preferably form a species chosen from the family consisting of Clupeidae and Engraulidae, more preferably chosen from the species herring (Clupea harengus) and anchoveta (Engraulis ringens) .
  • the oil has an omega-3-PUFA content of less than 14 % before enrichment of the acylglycerols with omega-3 PUFA and has an omega-3-PUFA content of more than 22 % after the enrichment.
  • the oil has a DHA content of less than 8 % before enrichment of the acylglycerols with omega-3 PUFA and has DHA content of more than 13 % after the enrichment.
  • the oil has an EPA content of less than 6 %before enrichment of the acylglycerols with omega-3 PUFA and has an EPA content of more than 8 %.
  • the DHA content comprised in the acylglycerols is increased by a factor of at least 1.9, preferably of at least 2.5 in a lipase-catalysed omega-3 concentration process, having one hydrolysis step and by a factor of at least 3.9 in a repeated hydrolysis.
  • the EPA content comprised in the acylglycerols is increased by a factor of at least 1.5 in a lipase-catalysed omega-3 concentration process, having one hydrolysis step and by a factor of at least 2 when the hydrolysis is repeated.
  • the content of polyunsaturated fatty acids comprised in the acylglycerols is increased by a factor of at least 1.7, preferably of at least 2.4 in a lipase-catalysed concentration process, having one hydrolysis step and by a factor of at least 3 in a repeated hydrolysis.
  • Another aspect of the present invention relates to the use of a processed marine oil according to any of preceding paragraphs as an ingredient for a feed, a functional feed, a health product, a cosmetic composition, or a pharmaceutical composition.
  • Figure 1 shows the time courses of hydrolysis reactions catalyzed by different lipases from Penicillium camembertii (PC), Rhizomucor javanicus (RJ), Rhizopus niveus (RN), Rhizopus delemar (RD), Burkholderia cepacia (BC), Rhizopus oryzae (RO), Candida rugosa (CR), and Rhizomucor miehei (RM).
  • PC Penicillium camembertii
  • RJ Rhizomucor javanicus
  • RN Rhizopus niveus
  • RD Rhizopus delemar
  • BC Burkholderia cepacia
  • RO Rhizopus oryzae
  • Candida rugosa CR
  • Rhizomucor miehei RM
  • Figure 2 shows changes in total omega-3 PUFA content related to the hydrolysis degree (HD, %) during hydrolysis catalyzed by lipases from Burkholderia cepacia (BC), Rhizopus oryzae (RO), and Candida rugosa (CR).
  • BC Burkholderia cepacia
  • RO Rhizopus oryzae
  • CR Candida rugosa
  • Figure 3 shows relationship between hydrolysis degree (HD, %) and hydrolysis resistant value (HRV, %) for (A) EPA, (B) DHA, and (C) OA using lipases from Burkholderia cepacia (BC), Rhizopus oryzae (RO), and Candida rugosa (CR).
  • Figure 4 shows the main effect of (A) temperature on total omega-3; (B) reaction time on EPA/DHA; (C) enzyme load (%) on OA/total omega-3 in the lipase-catalysed hydrolysis of salmon oil catalysed by a lipase from Candida rugosa.
  • DHA shows relationship between hydrolysis degree (HD, %) and hydrolysis resistant value (HRV, %) for (A) EPA, (B) DHA, and (C) OA using lipases from Burkholderia cepacia (BC), Rhizopus oryzae (RO), and Candida rugosa (CR).
  • Figure 4 shows the main effect of (A) temperature on total
  • docosahexaenoic acid EPA, eicosapentaenoic acid
  • OA oleic acid
  • Figure 5 shows the response surface plot demonstrating the effect of temperature and reaction time on the total omega-3 response at a fixed enzyme load at 3% while the ratio between oil and water phases was equal to 1 (lipase from Candida rugosa).
  • Figure 6 shows the response surface plot demonstrating the effect of enzyme load and water-to-oil ratio on the EPA/DHA response at a fixed temperature of 30°C and reaction time of 4 h (lipase from Candida rugosa).
  • Figure 7 shows the response surface plot demonstrating the effect of enzyme load and water-to-oil ratio on the OA/total omega-3 response at a fixed temperature of 30°C and time of 4 h (lipase from Candida rugosa).
  • Figure 8 shows the time course of the change in total omega 3 PUFA content during hydrolysis of different fish oils (lipase from Candida rugosa).
  • Figure 9 shows the lipid class compositions of product and residue after applying the process for large scale hydrolysis followed by short path distillation of the hydrolysed product.
  • MG monoacylglycerols
  • DG diacylglycerols
  • TG triacylglycerols
  • FFA free fatty acids (lipase from Candida rugosa).
  • Figure 10 shows a comparison of the fatty acid profiles of substrate, residue and distillate fractions after enzymatic hydrolysis and short path distillation (lipase from Candida rugosa).
  • Figure 11 shows the changes in fatty acid content of the acylglycerol ( ⁇ , o) and FFA (a, ⁇ ) fractions obtained by single step (filled symbols) and repeated (non-filled symbols) hydrolysis throughout the reactions (lipase from Candida rugosa).
  • the lipase from Candida rugosa being the one with the best hydrolysis result in experiment 1 was chosen as an example candidate from this group for the optimization of the process conditions for this group of lipases with special focus on large scale applications (experiments 2-5).
  • the hydrolyzed fatty acids are released as free fatty acids, which eventually can be separated from the acylglycerol fraction comprising PUFA by different fractionating/-separation methods.
  • Upgrading of farmed salmon fish oil obtained from by-products was carried out by lipase-catalyzed hydrolysis to increase omega-3 polyunsaturated fatty acids (PUFA) content.
  • the lipases tested were lipases which hydrolyze saturated and monounsaturated fatty acids selectively, and discriminate against omega-3 polyunsaturated fatty acids.
  • Salmon oil from by-products of farmed salmon was produced according to the enzymatic process by Sorensen et al. (2004) involving hydrolysis of by-products by a protease enzyme.
  • Rhizopus delemar (RD; 1092.5 U/g), Burkholderia cepacia (BC; previously known as Pseudomonas cepacia; 705.1 U/g), and Rhizopus oryzae (RO; 914.7 U/g) were from Amano (Virginia, VA, USA) while Candida rugosa lipase (CR; previously known as Candida cylindracea; 1489.2 U/g) was purchased from Fluka (Buchs, Switzerland). Immobilized lipase from Rhizomucor miehei (RM; 282 U/g) was provided by
  • Lipid class analysis by TLC-FID Samples were analyzed by thin layer chromatography coupled with a flame ionization detector (latroscan MK-6 s, Bechenheim, Germany). Aliquots of 20 ⁇ _ were dissolved in 0.8 ml_ of chloroform/methanol mixture (2:1 , v/v), and 1 ⁇ _ of diluted sample was spotted onto silica-coated Chromarod quartz rods by a semiautomatic sample spotter. Samples were developed with the developing system of n-hexane, diethyl ether, and acetic acid (45:25:1 , v/v/v). The rods were dried for 2 min at 120°C prior to analysis. The area percentages of TG, DG, MG, and FFA were used for the calculation of product yields. Hydrolysis degree is defined as (100-TG), %. All analyses were done in duplicate. The adopted values are the means at the 95% confidence limit.
  • the FA compositions of fractions were investigated by gas chromatography (Thermo Trace GC Ultra, USA) equipped with an autosampler, a flame ionization detector and a Omegawax 250 fused silica capillary column (30 m x 0.25 mm x 0.25 ⁇ film thickness; Supelco, Bellefonte, PA, USA). Helium was used as the carrier gas with a flow rate of 1 mL/min.
  • a temperature program was set as follows: increasing from 170°C to 215°C at a rate of 1 °C/min, held at 215°C for 15 min.
  • the injector and detector temperatures were set at 250°C and 270°C, respectively.
  • FAs were identified by comparing their retention times with standard mixtures and expressed as wt% after correction for detector response factors.
  • the time course of the hydrolysis for different lipases is shown in figure 1.
  • the hydrolysis reaction reached equilibrium for all the lipases tested after 12 to 24 h.
  • the HD (hydrolysis degree) values obtained were 1 1.97%, 19.68%, and 9.42% for the PC, RJ, and RN lipases respectively.
  • the HD was 35.33%, 47.24%, 47.33%, and 54.97%, respectively, by the lipases from CR, RM, BC, and RO after the first 30 min.
  • CR lipase had the highest increase in omega-3 PUFA content. The value was increased by approximately 50% (20.54%) after 2 h and reached to 27.81 % at the end of the 24 h period.
  • CR lipase is previously recognized as an enzyme from this group which has a high hydrolysis efficiency and discrimination against EPA and DHA. This lipase had no positional specificity, hydrolyzing FAs at all sn- positions randomly even at HD levels as low as 20%. On the other hand, it had a strict acyl chain specificity, resulting in selective hydrolysis of SFA and MUFA at a much higher rate compared to omega-3 PUFA.
  • Table 1 FA content in the acylglycerol fraction before the start of the hydrolysis (0 h) and after 24 h of hydrolysis catalyzed by selected lipases (RO, BC and CR).
  • Figure 2 shows the changes in total omega-3 PUFA content related to HD during hydrolysis catalyzed by lipases CR, BC and RO.
  • the changes in omega-3 PUFA content were significantly different although the HD values were similar for all the lipases at the end of 24 h.
  • CR lipase resulted in constant increase in total omega-3 PUFA content with increasing HD above 40%.
  • the other lipases showed a slight decrease in total omega-3 PUFA above 50% of HD.
  • the hydrolysis resistant value (HRV) was calculated according to Tanaka et al.
  • HRV (%) ⁇ (100 x GC a - B x GC b ) ⁇ (100 x GC a ) ⁇ x 100
  • GC a is the content of each FA in the original oil
  • GCb is the content of each FA in the FFA fraction of the product, both of which were measured by GC (wt %)
  • B is the ratio of FFA in the hydrolyzed oil, measured by TLC-FID (vol %).
  • a high HRV indicates a high resistance of the FA of interest to hydrolysis.
  • the concentration of omega-3 PUFA in salmon oil depends on one hand on the discrimination of the lipase against EPA and DHA, as well as the selective hydrolysis of monounsaturated fatty acids such as for OA, the target FA.
  • the efficiency of the lipases depends on the other hand on the chosen reaction conditions as well as the combination of reaction conditions which is considered to be important for the efficiency of the lipase catalysed concentrating process of oils.
  • Salmon oil from by-products of farmed salmon was produced according to the patented enzymatic process by Sorensen et al. (2004; EP 1 575 374 B1 ) involving hydrolysis of by-products by a protease enzyme.
  • the major FA found in the substrate was oleic acid (OA) with a share of 35.51 %, while the omega-3 PUFA content was as follows: 4.8% EPA, 2.04% docosapentaenoic acid (DPA), 6.93% DHA.
  • DPA docosapentaenoic acid
  • Lipase from Candida rugosa 64000 U/g was donated by Meito Sangyo Co., Ltd. (Tokyo, Japan).
  • Fatty acid methyl ester standard was purchased from Nu-Chek- Prep (Elysian, MN, USA). All other reagents and solvents used were from Sigma- Aldrich Co. (St. Louis, MO, USA) and of chromatographic grade.
  • CCD Central composite design
  • acylglycerol fraction consisting of triacylglycerols (TG), diacylglycerols (DG) and monoacylglycerols (MG), was extracted twice by 2 mL hexane after saponification of free fatty acids (FFA) by adding 0.5 mL of 0.5 M ethanolic KOH.
  • the ethanolic water phase was acidified by 0.3 mL of 2 M HCI, and FFA fraction was extracted by hexane similarly. Fractions were methylated according to the AOCS method Ce-1 b (2007).
  • the FA compositions of fractions were investigated by gas chromatography (GC) (Thermo Trace GC Ultra, USA) equipped with an autosampler, a flame ionization detector and a Omegawax 250 fused silica capillary column (30 m x 0.25 mm x 0.25 ⁇ film thickness; Supelco, Bellefonte, PA, USA). Helium was used as the carrier gas with a flow rate of 1 mL/min.
  • a temperature program was set as follows: increasing from 170°C to 215°C at a rate of 1 °C/min, held at 215°C for 15 min.
  • the injector and detector temperatures were set at 250°C and 270°C, respectively.
  • FAs were identified by comparing their retention times with standard mixtures and expressed as wt% after correction for detector response factors.
  • the data were analyzed by means of RSM using Modde 8.0.2.
  • Dependent variables were chosen to be total content of EPA, DPA and DHA in the acylglycerol fraction (total omega-3), the ratio of EPA to DHA in the acylglycerol fraction (EPA/DHA), and the ratio of OA to the sum of EPA, DPA and DHA in the FFA fraction (OA/total omega-3).
  • the responses were first fitted to factors by multiple regressions, and then the models generated were used to evaluate the effects of various factors.
  • the first- and second-order coefficients were generated by regression analysis (Table 3).
  • the accuracy of the models was evaluated by coefficient of determination (R 2 ), absolute average deviation (AAD) and a test for lack of fit from analysis of variances
  • Yi.obs and yi,pr are the observed and predicted responses, respectively, and p is the number of experimental run.
  • R 2 must be close to 1.0 and the AAD between the predicted and observed data must be as small as possible.
  • Quadratic polynomial regression models were assumed for predicting the responses.
  • the model proposed for each response was
  • Y is the response (total omega-3; EPA/DHA; OA/total omega-3), /3 ⁇ 4 is the intercept, ⁇ , is the first-order model coefficient, ⁇ trimming is the quadratic coefficient for the variable, j8,y is the interaction coefficient for the interaction of variables / ' and j, X, and Xj are the independent variables (Te, t, En, W r ).
  • Five additional reactions were performed at the optimized levels to verify the models by chi-square ( ⁇ 2 ) test.
  • the main objective of the study was to concentrate/enrich the omega-3 PUFA content in the acylglycerol fraction by releasing the rest of the FAs through lipase- catalyzed hydrolysis.
  • Responses obtained from the experimental design are given in Table 2.
  • R 2 and AAD values of the model generated based on total omega-3 response were 0.94 and 2.76, respectively.
  • ANOVA also revealed that the probability for the regression of the model was significant (P ⁇ 0.001 ), confirming that the model was statistically good, and it had no lack of fit (P>0.05). According to the regression coefficients (Table 3), the most significant linear effect at the significance level of 95% was that of temperature.
  • FIG. 4A shows the main effect of temperature on the total omega-3 content.
  • Temperature plays an important role in enzymatic reactions, mainly by determining the reaction rate. Although increased temperature enhances the hydrolytic rate, it can also lead to thermal deactivation of lipase. Moreover, increased temperature results in oxidation of PUFA to a higher extent, which could also be an explanation of the lower rate of increase in the total omega-3 PUFA content at elevated temperatures.
  • Figure 4B shows the main effect of time on EPA/DHA response. Short reaction time was not sufficient to have a reasonable hydrolysis degree. After 4 h of reaction (reaction 9 in Table 2), 82.3% of the initial TAG was unhydrolyzed (data not shown), which explains why EPA/DHA value (0.63) was closer to the original value, and in fact, was the highest value obtained. It was suggested that hydrolysis reaction catalyzed by Candida rugosa lipase took place in two steps: TG molecules without DHA were hydrolyzed at the earlier stages of the reaction. As the reaction progressed, DHA containing-TG molecules are hydrolyzed as well which results in faster clearance of EPA compared to DHA.
  • Oleic acid was the dominant FA in the salmon oil composition, with 35.51 % share, due to the feed given in the salmon farm.
  • OA was chosen as the target FA to be removed from the oil.
  • Monitoring the ratio between OA and total omega-3 PUFA released to the FFA fraction by hydrolysis enabled to compare the selectivity of the lipase towards these FAs.
  • Responses obtained from the experimental design are given in Table 2.
  • R 2 and AAD values of the model were 0.9 and 4.73, respectively.
  • ANOVA revealed that the probability for the regression of the model were significant (P ⁇ 0.001 ), confirming that the model was statistically good, and it had no lack of fit at 95% level of significance.
  • omega-3 content of the substrate could be increased above 32.5% with a temperature of 40-50°C by a 4-h reaction while enzyme load was 3% and the weight ratio between oil and water phases was equal to 1.
  • Figure 6 shows the response surface plot indicating the effect of enzyme load and water-to-oil ratio on the EPA/DHA level while the other two factors are fixed at their lowest levels, since increasing both of these factors resulted in decreased response. None of the combinations in the design ranges resulted in a similar EPA/DHA value with that of salmon oil, which was 0.7. Hydrolysis degree increases with increased water-to-oil ratio, due to the available interphase between the substrates. EPA is more prone to hydrolysis compared to DHA, which was depicted by decreased EPA/DHA level at water-to-oil ratios higher than 3. However, since the linear effect of enzyme load is much more significant than that of water-to-oil ratio, EPA/DHA response was highly dependent on the amount of enzyme.
  • EPA/DHA level could be maintained above 0.55 with 2-5 water-to-oil ratio and 3-6% enzyme load by a 4-h reaction at 30°C.
  • the highest level for the response was obtained when the enzyme load was 3%, and decreased as the amount of enzyme increased. This result is due to the high availability of enzyme in the medium, and thus, decreased selectivity of the hydrolytic reaction, resulting in hydrolysis of omega-3 PUFA as well as OA.
  • the FA selectivity of the Candida rugosa lipase was believed to be interfered by increased FFA content in the medium at elevated content of water, as a result of the increased hydrolytic rate as well as the
  • Candida rugosa lipase as an example representing lipases which selectively hydrolyse saturated and monounsaturated fatty acids bound to said acylglycerols and discriminate in its hydrolytic activity against omega-3 polyunsaturated fatty acids, significantly increased the content of omega-3 PUFA in the substrate.
  • omega-3 PUFA As well as the ratio between them (EPA/DHA) in the product and the ratio of OA to total omega-3 PUFA released by hydrolysis, most optimum conditions were selected as follows: 45°C of temperature, 4 h of time, 3% of lipase (based on oil amount), 3.16-fold water (w/w, based on oil amount).
  • Total omega-3 PUFA content of the product obtained at the given conditions was increased from 13.77% to 33.01%.
  • EPA/DHA ratio was acceptable (0.48), while the ratio of OA to total omega-3 PUFA in the FFA fraction was significantly high (9.53).
  • -incubation temperature between 30 and 50°C, preferably between 40 and 50°C, most preferred 45°C .
  • reaction time should be at least 2 hours, preferably in the range of 2-8 hours.
  • the lipase is added in a concentration of at least 3 % by weight based on the amount of oil
  • the incubation temperature is about 45 °C
  • the incubation time is between 2 hours and 4 hours.
  • the lipase is from Pseudomonas, Candida, Rhizopus, Rhizomucor. Particularly preferred lipases are from Candida rugosa, Burkholderia cepacia and Rhizopus oryzae, however most preferred is the lipase from Candida rugosa.
  • the examined lipases are all belonging to the group of lipases which selectively hydrolyse saturated and monounsaturated fatty acids bound to said acylglycerols and discriminate in their hydrolytic activity against omega-3 polyunsaturated fatty acids.
  • the optimization study has been based on the lipase from Candida rugosa as the most promising representative of the group, it is assumed that the optimized conditions will also be beneficial in lipase-catalyzed omega-3 fatty acid enrichment processes with the other lipases belonging to that group.
  • the process is especially suitable when used in an industrial applicable large-scale process due to its mild and at the same time cost and time-efficient features.
  • Salmon oil obtained according to the enzymatic process as described by Sorensen et al. (2004) involving hydrolysis of by-products by a protease enzyme, pelagic oil obtained from herring (Clupea harengus) and two types of commercially available fish oils (from EPAX, Norway) were hydrolysed using Candida rugosa lipase according to the preferred conditions of experiment 2 (3% of lipase, incubation temperature of 45 °C, water-to-oil ratio of about 3.16:1 (v/w), and incubation time of 4 hours).
  • Fatty acid compositions of substrates are given in Table 5.
  • the EPA/DHA ratio in the salmon oil was 0.67 initially (Table 8). The ratio decreased to 0.44 at the end of the 4 h lasting reaction. The obtained EPA/DHA ratio in the present invention is considered satisfactory for the intended purpose and final product in spite of the short reaction time applied in the present invention.
  • the only substrate with an initial EPA/DHA ratio higher than 1 was EPAX 3000, which did not decrease significantly after 4 h of reaction.
  • the concentrating process of the present invention resulted in an enrichment factor of about 2.4 in the salmon oil for omega-3 PUFA, about 1.8 for the pelagic oil and 1.4 for the commercial EPAX 3000 oil.
  • This further supports the suitability of the process for the enrichment of marine oils, especially of the salmon oil and the pelagic oil with a reduced content of omega-3 PUFA.
  • Due to the high efficiency of the process it is assumed that the process of the present invention may also be used for oils having an even lower content of omega-3 PUFA than in the present experiment such as for oils from farmed salmon fed very low amounts of omega-3 fatty acids. It is therefore preferred that also an oil of farmed fish having a very low content of PUFA can be used.
  • Salmon oil from by-products of farmed salmon was produced according to the patented enzymatic process by Sorensen et al (2004) involving hydrolysis of byproducts by a protease enzyme. As in the previous experiments a lipase from
  • Candida rugosa lipase 64000 U/g was used.
  • Lipid class analysis by TLC-FID Lipid class analysis by TLC-FID was carried out as described in experiment 3.
  • a repeated hydrolysis was performed on the residue obtained from short path distillation.
  • the second hydrolysis was carried out with the same enzyme as in the first hydrolysis.
  • Three grams of the residue obtained from Experiment 4 was subjected to a further hydrolysis under the same reaction conditions as in the first hydrolysis. The reaction was continued until the FFA level was approximately 40%.
  • Figure 11 compares the changes in FA content of the acylglycerols and FFA fractions obtained by single step and repeated hydrolysis throughout the reactions.
  • a comparison of contents of major FAs in substrate and hydrolysis products are given in Table 10.
  • the total omega-3 PUFA content was further concentrated to 50.58% by repeated hydrolysis.
  • the loss of omega-3 PUFA to the FFA fraction was increased as well ( Figure 1 1 ).
  • Due to the reduced availability of SFA and MUFA DHA was hydrolyzed to a certain degree in the second round of hydrolysis, which resulted in the increased omega-3 PUFA content in the FFA fraction obtained after repeated hydrolysis.
  • the concentration achieved was, however, more than 3 times of the original level.
  • the added value of the product was surprisingly further improved by the repeated hydrolysis approach even though using the same enzyme as in the first hydrolysis.
  • Total omega-3 PUFA - refers to the sum of EPA, DPA and DHA
  • Fish oil - by fish oil is meant an oil originating from fish comprising omega-3 polyunsaturated fatty acids such as EPA and/or DHA and/or DPA.
  • Pelagic oil - by pelagic oil is meant an oil obtained from a pelagic fish such as from herring (Clupea harengus) or anchoveta (Engraulis ringens), wherein the raw material (e.g. viscera after filleting, whole fish) is processed by a commonly known fish meal/ fish oil production method without enzymatic treatment.
  • Marine oil - by marine oil is meant a fish oil or oils produced from other animals or plants or oils produced by microorganisms such as bacteria or algae, which comprise polyunsaturated fatty acids such as EPA and/or DHA and/or DPA.
  • Marine/Fish oil concentrate or processed marine oil - by an oil concentrate or in the context of the present invention by a processed marine oil is meant an oil wherein the amount of omega-3 fatty acids in the acylglycerol fraction of the oil has been increased (enriched, up-graded, up-concentrated) in relation to the total amount of fatty acids comprised in said acylglycerol fraction by means of a
  • Enrichment of an oil with omega-3 PUFA /concentration of omega-3 PUFA in an oil - by enrichment of an oil with omega-3 PUFA or concentrating of omega-3 PUFA in an oil is meant that the amount of omega-3 PUFA or of a specific omega-3 fatty acid such as DHA or EPA is increased in the acylglycerol fraction of the oil in relation to the total amount of fatty acids comprised in said acylglycerol fraction by means of a enrichment/concentrating process.
  • Lipase which discriminates in its activity against certain fatty acids - by discriminating in its activity against certain fatty acids such as omega-3 fatty acids is meant that the lipase has a negative selectivity (or lower substrate affinity) towards these fatty acids and preferably hydrolyzes other fatty acids bound in acylglycerols e.g. those which are not omega-3 fatty acids such as saturated and monounsaturated fatty acids.

Abstract

La présente invention concerne un procédé enzymatique pour l'enrichissement de la teneur en PUFA oméga 3 dans la fraction acylglycérol d'huiles d'animaux marins adaptée à une application à grande échelle, ainsi que des huiles d'animaux marins enrichies en acides gras oméga 3 polyinsaturés et leur utilisation.
PCT/NO2011/000354 2010-12-23 2011-12-22 Enrichissement d'huiles d'animaux marins en acides gras oméga 3 polyinsaturés par une hydrolyse catalysée par une lipase WO2012087153A1 (fr)

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EP2758480A1 (fr) * 2011-09-19 2014-07-30 Bomi P. Framroze Huile comestible ayant une concentration élevée d'acides gras polyinsaturés
US9050309B2 (en) 2012-01-06 2015-06-09 Omthera Pharmaceuticals, Inc. DPA-enriched compositions of omega-3 polyunsaturated fatty acids in free acid form
US9492545B2 (en) 2012-05-07 2016-11-15 Omthera Pharmaceuticals Inc. Compositions of statins and omega-3 fatty acids
CN109053420A (zh) * 2018-08-21 2018-12-21 浙江海洋大学 一种从桡足类中提取dha和epa的方法
WO2019038783A1 (fr) * 2017-08-22 2019-02-28 Praj Industries Limited Préparation de glycérides enrichis en epa et dha
CN110684906A (zh) * 2018-07-05 2020-01-14 南京长江江宇石化有限公司 过氧化法制取pomtbe中含钼残液的回收再利用方法
KR102085775B1 (ko) * 2018-12-14 2020-04-24 송광수 참치 껍질로부터 dha를 분리 및 정제하는 방법
US20210315941A1 (en) * 2020-04-07 2021-10-14 Hofseth Biocare Asa Respiratory treatments using salmonid oil compositions
EP3848465A4 (fr) * 2018-09-04 2022-06-29 Nippon Suisan Kaisha, Ltd. Procédé de fabrication de glycéride à teneur en acide docosahexaénoïque mettant en oeuvre une réaction d'hydrolyse par lipase
US11840714B2 (en) 2018-09-04 2023-12-12 Nissui Corporation Enriching DHA in glyceride fractions

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EP2758480A4 (fr) * 2011-09-19 2015-01-28 Hofseth Biocare Asa Huile comestible ayant une concentration élevée d'acides gras polyinsaturés
EP2758480A1 (fr) * 2011-09-19 2014-07-30 Bomi P. Framroze Huile comestible ayant une concentration élevée d'acides gras polyinsaturés
US9050309B2 (en) 2012-01-06 2015-06-09 Omthera Pharmaceuticals, Inc. DPA-enriched compositions of omega-3 polyunsaturated fatty acids in free acid form
US9050308B2 (en) 2012-01-06 2015-06-09 Omthera Pharmaceuticals, Inc. DPA-enriched compositions of omega-3 polyunsaturated fatty acids in free acid form
US10117844B2 (en) 2012-01-06 2018-11-06 Omthera Pharmaceuticals, Inc. DPA-enriched compositions of omega-3 polyunsaturated fatty acids in free acid form
US9492545B2 (en) 2012-05-07 2016-11-15 Omthera Pharmaceuticals Inc. Compositions of statins and omega-3 fatty acids
US11345939B2 (en) 2017-08-22 2022-05-31 Praj Industries Limited Preparation of EPA and DHA enriched glycerides
WO2019038783A1 (fr) * 2017-08-22 2019-02-28 Praj Industries Limited Préparation de glycérides enrichis en epa et dha
CN110684906A (zh) * 2018-07-05 2020-01-14 南京长江江宇石化有限公司 过氧化法制取pomtbe中含钼残液的回收再利用方法
CN109053420A (zh) * 2018-08-21 2018-12-21 浙江海洋大学 一种从桡足类中提取dha和epa的方法
CN109053420B (zh) * 2018-08-21 2021-03-16 浙江海洋大学 一种从桡足类中提取dha和epa的方法
EP3848465A4 (fr) * 2018-09-04 2022-06-29 Nippon Suisan Kaisha, Ltd. Procédé de fabrication de glycéride à teneur en acide docosahexaénoïque mettant en oeuvre une réaction d'hydrolyse par lipase
US11840714B2 (en) 2018-09-04 2023-12-12 Nissui Corporation Enriching DHA in glyceride fractions
KR102085775B1 (ko) * 2018-12-14 2020-04-24 송광수 참치 껍질로부터 dha를 분리 및 정제하는 방법
US20210315941A1 (en) * 2020-04-07 2021-10-14 Hofseth Biocare Asa Respiratory treatments using salmonid oil compositions

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